Mark J. Zoller

Oligonucleotide-Directed Mutagenesis of DNA Fragments Cloned into M13 Vectors

Publisher Summary Oligonucleotide-directed in vitro mutagenesis provides a means to alter a defined site within a region of cloned DNA. This powerful technique has many far-reaching applications, from the definition of functional DNA sequences to the construction of new proteins. The method stemmed from the combination of a number of recent discoveries and observations about nucleic acids but the basic principle involves the enzymic extension by E. coli DNA polymerase of an oligonucleotide primer hybridized to a single-stranded circular template. More recently, phage M13 and M13 derived vectors have been used in a number of oligonucleotide mutagenesis experiments. Vectors derived from single-stranded phage, such as M13 and fd, are more convenient for oligonucleotide-directed mutagenesis than double-stranded vectors, because isolation of pure single-stranded circular template DNA in these systems is quite simple. This chapter describes a mutagenesis procedure that is simple, versatile, and efficient. The chapter also explains the targets and vehicles utilized in a number of recent mutagenesis experiments.

Mutagenesis of Fujinami Sarcoma Virus: Evidence that tyrosine phosphorylation of P130gag-fps modulates its biological activity

The 130 kd transforming protein of Fujinami sarcoma virus (FSV P130gag -fps) possesses a tyrosine-specific protein kinase activity and is itself phosphorylated at several tyrosine and serine residues in FSV-transformed cells. We have used oligonucleotide-directed mutagenesis of the FSV genome to change the TAT codon for tyrosine (1073), the major site of P130gag -fps phosphorylation, to a TTT codon for phenylalanine that cannot be phosphorylated. This mutant FSV induces the transformation of rat-2 cells but with a long latent period as compared with wild-type FSV. The P130gag -fps protein encoded by the mutant retains the ability to phosphorylate tyrosine, but is five times less active as a kinase in vitro than wild-type FSV P130gag -fps. These data indicate that tyrosine phosphorylation stimulates the biochemical and biological activities of FSV P130gag -fps, and they set a precedent for the ability of this amino acid modification to modulate protein function.

30 – Oligonucleotide-Directed Mutagenesis of DNA Fragments Cloned into M13 Vectors

Publisher Summary nThis chapter discusses the oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors. The isolation and sequencing of genes has been a major focus of biological research for almost a decade. The emphasis has at present turned toward the identification of functional regions encoded within DNA sequences. The oligonucleotide is designed so that the mismatch(es) is located near the middle of the molecule. This is especially important if the oligomer will be used as a probe to screen for the mutant. Short oligonucleotides can be synthesized in vitro by either enzymic or chemical means. The mutagenic oligonucleotide and single-stranded template DNA are annealed and extended by DNA polymerase using the conditions determined in the preliminary tests. The CC-DNA obtained from the sucrose gradient is a heteroduplex consisting of one wild-type strand and one mutant strand.